This project is aimed towards understanding the mechanisms which mediate and regulate calcium signaling in salivary gland cells. Neurotransmitter stimulation of fluid secretion in salivary glands is mediated via a biphasic elevation in cytosolic [Ca-2+]; an initial transient increase due to internal release and a latter sustained increase due to Ca-2+ influx. Sustained fluid secretion is directly dependent upon the sustained elevation of [Ca-2+] and thus on neurotransmitter-stimulated calcium influx. In the past 8-10 years, our efforts have been focused on the neurotransmitter-stimulated calcium influx mechanism in salivary gland cells. Recent data from our laboratory and others demonstrate that two types of calcium entry mechanisms can contribute to this calcium signal; store-operated calcium entry (which is activated by the depletion of calcium in the intracellular calcium store) and receptor-or second messenger-operated calcium entry (which is activated directly by receptor signaling via second messengers such as diacylglycerol that are generated in response to neurotransmitter-stimulated phosphatidyl inositol bisphosphate hydrolysis). These calcium entry pathways are ubiquitously present in excitable and non-excitable cells and critically affect a number cellular functions. The molecular components or regulatory mechanism(s) of these calcium entry pathways have not yet been established in any cell type. Members of the transient receptor potential (TRPC) family of ion channel proteins have been proposed as molecular components of the neurotransmitter-activated calcium influx channels. All TRPCs have the ability to be activated by agonist-stimulation of phosphatidyl inositol bisphosphate hydrolysis and contribute to both types of calcium entry. The physiological function(s) and regulation of the presently identified TRPCs have not yet been fully established. Further, how they contribute to both store-operated and store-independent calcium entry pathways is not known. By expressing TRPC1 in vivo in rat SMG by using an adenovirus encoding hTrp1 (AdHA-hTrp1) and by examining the role of native and mutant TRPC1 in the HSG cell line, we had previously reported that TRPC1 is involved in the regulation of store-operated calcium influx in salivary gland cells. In the past year we have continued to characterize agonist-regulated calcium entry and identify the role of TRPC channels in this mechanism in salivary gland cells. Our previous studies have been largely carried out with HSG cells in which we have demonstrated conclusively that TRPC1 is the primary store-operated calcium entry channel component. Towards characterizing this channel in other salivary gland cells, we have now studied calcium entry in HSY (human parotid gland cell line) cells. Our previous results demonstrated that distinct store-operated calcium channels are present in HSG (human submandibular gland cell line) and HSY cells. Both of these channels were different from the channel in rat basophilic leukemia cells, the components of which are presently unknown. Although the physiological relevance of these different SOCE channels is not presently clear, we have now examined the molecular components of the channel in HSY cells. Interestingly, while the HSG channel appears to primarily depend on TRPC1, the HSY channel appears to be formed by the co-assembly of TRPC1 and TRPC3. Further, TRPC1-TRPC3 interactions are mediated via their N-terminal domains. Expression of the N-terminal domain of either TRPC1 or TRPC3, or a dominant negative mutant of TRPC1, disrupts calcium entry in HSY cells. This effect on calcium entry is due to the competetive interaction of the expressed proteins with endogenous TRPC proteins and disruption of channel assembly. Thus, we propose that TRPCs can assemble as homomers or heteromers to form store-operated calcium channels and that the channel properties are defined by the specific TRPC components that are involved. We have previously hypothesized that the type of channel formed in a cell will depend on the physiological function it serves in that particular cell. To further assess the physiological relevance of TRPC channels in salivary glands we have examined their routing in polarized epithelial cells. We have established stable TRPC expression in MDCK (canine kidney cell line) and rat salivary epithelial (SMIE) cells, both of which form high resistance monolayers when cultured on Transwell filters. We have observed that TRPCs have distinct cellular localization. TRPC3 is apically localized, TRPC1, TRPC5, and TRPC2 are basally localized while TRPC6 is found in both apical and basal regions of the cell. Further, endogenous TRPC3, TRPC1, and TRPC6 were found at the same locale. We have also studied the regulation of the TRP channels in these cellular regions. Consistent with previous reports, calcium signaling proteins were also predominantly localized in the apical region of these cells. Further, TRPC3 was assembled in a complex with TRPC6, but not TRPC1, and key calcium signaling proteins like inositol trisphosphate receptor, G-proteins, and phospholipase C. Importantly, we showed that TRPC3/TRPC6 channels can mediate apical calcium uptake and transepithelial calcium transport in polarized epithelial cells. These data demonstrate a novel role for TRPC3/TRPC6 channels; i.e. agonist-stimulated apical calcium uptake. Consistent with this, we detected localization of TRPC3 and TRPC6 in the apical regions of rat submandibular gland and kidney ducts. Further, addition of OAG (a diacylglycerol analogue) induced calcium entry in isolated rat submandibular gland ducts. These data indicate that non-store-operated channels are formed by TRPC3-TRPC6 interaction in the luminal membrane of salivary gland ducts. Studies are ongoing to determine how these apically localized channels are regulated and what is their physiological function. In another study members of SPS closely collaborated with Dr. Barker?s lab (NINDS) to demonstrate that TRPC1 regulates fibroblast growth factor (FGF) receptor-mediated growth of rat embryonic neuronal stem cells. We performed all the electrophysiology as well as studies showing the interaction and localization of the FGF receptor as well as TRPC1. These data add to the emerging concept that TRPC channels contribute to critical cellular functions such as secretion, proliferation, and cell death. In the coming fiscal year we will continue our studies along these directions. A major focus will be directed towards determining novel TRPC interacting proteins to help us to understand their function and regulation. We will also continue to study the trafficking of TRPC channels and the mechanisms involved in their assembly and multimerization. Studies will also be directed towards identifying specific channels generated by different TRPC combinations. In new studies that are ongoing we are also looking at the role of osmosensing TRPV channels in the regulation of water permeability and cell volume in salivary gland cells. Another study is directed towards identifying the internal calcium store that is coupled to the plasma membrane channel and the mechanism relaying signals between these two cellular membranes. We further plan to use various mouse models as well as in vivo adenoviral directed expression of TRPC channels or RNAi to further establish the role of these channels in salivary gland function. We believe that together our studies will provide substantial information about the mechanisms regulating salivary gland fluid secretion.